Malaria is an insect-borne disease that is widespread in tropical and subtropical regions, including parts of Asia, Africa and the Americas. As well as its human cost, malaria also has an economic impact particularly in developing countries where the disease is endemic. Malaria infection rates are also rising in industrial countries, such as the USA and Canada, due to increased international travel, movement of refugees, immigration and migrant workers.
Four parasites are thought to be responsible for malaria: plasmodium falciparum, plasmodium vivax, plasmodium ovale and plasmodium malariae. The most common and serious form of the disease is caused by plasmodium falciparum. Malaria parasites are transmitted by female mosquitoes when they bite a victim. The parasites multiply within the victim's red blood cells causing symptoms such as fever, shivering, vomiting, anaemia, convulsions, and eventually coma and death if left untreated. At the early activated (trophozoite) stage, the malarial parasites are inside red blood cells and digest the haemoglobin of the red blood cells to synthesize amino acids to multiply. Heme is produced as a metabolic product of the degradation of haemoglobin and as the heme exhibits a strong cytotoxic effect to the malarial parasite, the malarial parasite converts the heme into haemozoin, a non-toxic polymer, which the malarial parasite deposits into food vacuoles in its body. The haemozoin is in the form of crystals comprising a hydrogen bonded chain of dimers formed by iron carboxylate bonds.
Due to the non-specific clinical symptoms of malaria, clinical diagnosis of malaria can be unreliable. Therefore, accurate diagnosis of malaria in a victim relies on testing the victim's blood. Blood testing methods for the detection of malaria include: 1) DNA/RNA staining methods e.g. Giemsa staining, Fluorescent staining, QBC®; 2) Antigen detection methods; 3) Automated malaria pigment detection methods; and 4) molecular methods (Hanscheid T. (1999) Diagnosis of malaria: a review of alternatives to conventional microscopy. Clin. Lab. Haem., 21, pp. 235-245).
Giemsa staining is the most commonly used detection method and involves the staining of blood films or smears to detect the DNA/RNA of the malaria parasite. The main advantage of this method is its low cost and the fact that parasitaemia levels as low as 0.0001% can be detected. The most significant drawback of this detection method is that it relies on the examination of blood samples by a technician using an optical microscope. Thus the proper training of technicians is required. Also, the quality and accuracy of the diagnosis relies on the skills and experience of the technician. Moreover, the process for identifying parasites by light microscopy is time consuming and multiple tests cannot be performed simultaneously. Furthermore, the processes of smearing, fixing and staining the blood prior to its analysis require specific equipment and reagents which are prone to degradation.
Another staining approach is fluorescence tagging of DNA/RNA. As red blood cells (mature mammalian erythrocytes) do not contain DNA whereas malaria parasites do, nucleotide specific fluorescent dyes can be used to detect the presence of the malaria parasite. The most common dye employed is acridine orange (AO), but the method still relies on the careful examination of blood samples as well as technician skill and generally does not give better results than the Giemsa staining method.
The DNA/RNA staining approach can be further refined using an AO coated capillary, termed QBC® (quantitative buffy coat), which is centrifuged and examined. Although this approach improves the sensitivity of the DNA/RNA staining approach and can be automated, it requires additional material, equipment, training, is more costly and is not more sensitive than the Giemsa staining method. Therefore, it has the same drawbacks as the Giemsa staining method.
Antigen detection methods detect the presence of proteins specific to malaria and are the most common approach to rapid malaria testing. There are three common formats: dipstick, cartridge and cassette. These methods are suitable for use in remote areas as they do not require specific operator training. Advantageously, many tests can be performed simultaneously. One such antigen detection method is HRP-2 available from Parasight® which detects the presence of a particular histidine-rich protein specific to the malaria parasite using a dipstick approach. The sensitivity of this method is comparable to the staining methods, and the time per test is shorter so many tests can be carried out simultaneously. However, antigen detection methods are generally more expensive than the staining methods. Also, the tests provide just a negative or a positive result and therefore more detailed examination is necessary to confirm the diagnosis. Furthermore, degradation of the test reagents of antigen detection methods are accelerated under high temperature and humidity conditions, such as those found in regions of the world where malaria is endemic.
Automated pigment detection methods typically use an automated haemotology analyzer and have the advantage of not requiring a skilled technician to prepare samples and operate a microscope. One such method involves the detection of an abnormal monocyte population using the Cell-Dyn™ 3500 analyzer (Abbott-Diagnostics, Maidenhead, UK). The main problems with the automated pigment detection methods are that they are unable to detect parasitaemia levels and that an abnormal monocyte population is not specific to malaria.
Molecular methods include a polymerase chain reaction method which uses specific parasite genetic sequences and is the most sensitive technique of all the above-mentioned techniques. However, this method is very time consuming and the high cost per test does not make it suitable for individual diagnosis.
Despite the fact that many techniques exist to detect haemozoin to attempt to diagnose malaria, there is a need for an improved system, method or device.